Synthetic substrates for thyroid oligosaccharide transferase. Effects of peptide chain length and modifications in the Asn-Xaa-Thr-region.
The N-glycosylation of proteins is initiated by transfer of preassembled oligosaccharides from lipid carriers to an asparagine residue in the sequence Asn-Xaa-Ser(Thr). Various synthetic peptides were previously shown to act as effective acceptors when supplied to microsomal membranes. The present work was undertaken to delineate further within such peptides the structural determinants required for the N-glycosylation process. Several new peptides were synthesized to evaluate the effects of the chain length and of the modification on the asparagine, Xaa or threonine residues: asparagine was replaced by glutamine, Xaa by proline or N-methylated alanine, threonine by an homoserine residue. Furthermore, both side chains of asparagine and threonine were simultaneously replaced by those of glutamine and homoserine respectively to restore a putative hydrogen bonding between the carbonyl and hydroxyl groups. The assays were performed with a solubilized form of the oligosaccharide transferase and the acceptor capacities of the peptides expressed by the ratio V/Km of their apparent kinetic parameters V and Km. Results showed that N-acetyl-tripeptide amides are excellent substrates for the enzyme. All modifications in the -Asn-Xaa-Thr- region resulted in a loss of acceptor capacity. The importance of the conformational aspect is discussed.
D000595:Amino Acid Sequence; D000818:Animals; D001216:Asparagine; D006602:Hexosyltransferases; D007202:Indicators and Reagents; D007700:Kinetics; D008565:Membrane Proteins; D009842:Oligopeptides / Q000138:chemical synthesis*; D013379:Substrate Specificity; D013912:Threonine; D013961:Thyroid Gland / Q000201:enzymology*; D014166:Transferases / Q000378:metabolism*
D007202:Indicators and Reagents; D008565:Membrane Proteins; D009842:Oligopeptides; D013912:Threonine; D001216:Asparagine; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase